Substance P (SP) may participate as a paracrine and/or autocrine factor in the regulation of anterior pituitary function. This project studied the effect of TRH on SP content and release from anterior pituitary and the role of SP in TRH-induced prolactin release. TRH (10(-7) M), but not vasoactive intestinal polypeptide (VIP), increased immunoreactive-SP (ir-SP) content and release from male rat anterior pituitary in vitro. An anti-prolactin serum also increased ir-SP release and content. In order to determine whether intrapituitary SP participates in TRH-induced prolactin release, anterior pituitaries were incubated with TRH (10(-7) M) and either WIN 62,577, a specific antagonist of the NK1 receptor, or a specific anti-SP serum. Both WIN 62,577 (10(-8) and 10(-7) M) and the anti-SP serum (1:250) blocked TRH-induced prolactin release. In order to study the interaction between TRH and SP on prolactin release, anterior pituitaries were incubated with either TRH (10(-7) M) or SP, or with both peptides. SP (10(-7) and 10(-6) M) by itself stimulated prolactin release. While 10(-7) M SP did not modify the TRH effect, 10(-6) M SP reduced TRH-stimulated prolactin release. SP (10(-5) M) alone failed to stimulate prolactin release and markedly decreased TRH-induced prolactin release. The present study shows that TRH stimulates ir-SP release and increases ir-SP content in the anterior pituitary. Our data also suggest that SP may act as a modulator of TRH effect on prolactin secretion by a paracrine mechanism.